The basic principle behind isoelectric focusing or focusing in a pH gradient is that a charged molecule will become immobilized in a electric field when it migrates to a position in the pH gradient that is equal to its isoelectric point (zero net charge). This process occurs independently of the initial location of a specific protein in the solution. It is the result of the disappearance of the effective electrical charge of the protein when migrating to the region where pH is equal to pI.
Various techniques for determining the isoelectric point of a protein have been described. Typically, the protein of interest is injected or administered directly into a gel containing a pH gradient, wherein the pH gradient is parallel to the direction of the electric field, and the protein can only be separated from other proteins by traveling uni-directionally through many different pH environments before reaching a pH environment that is equivalent to its isoelectric point. These techniques suffer from the disadvantages that (1) they require a relatively long time to separate the protein because the velocity of the fraction tends to zero asymptotically; (2) they require relatively high voltages (typically 1000V and higher), and (3) they require a cooling mechanism. Traditional IEF methods are labor intensive, time consuming, non-standardized, expensive and not sensitive. Another practical limitation of traditional isoelectric focusing gels is that it is difficult to manufacture gels having incrementally small pH changes within a pH gradient to improve the linear dispersion of the proteins.
Two dimensional analysis of proteins that use the above described isoelectric focusing step suffer from the same problems. For example, Zuo et al., (2000) Analytical Biochemistry 284:266–278, describe the separation of proteins based on their isoelectric point by unidirectional travel through a pH range followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Becker, et al., (1998) J. Micromech. Microeng. 8:24–28 suggests the unidirectional travel of proteins through a pH range followed by a second dimension separation on a planar chip. See also, U.S. Pat. No. 6,254,754 (Ross).
Because of these limitations, only certain cell, lane, and matrix designs and orientations of the cells, lanes, and matrixes in a chamber, and certain systems for one and two dimensional analysis are possible thereby limiting the development of faster, more sensitive, more accurate, more flexible and less expensive methods for one and two dimensional analyses of samples, including automated, high throughput analysis systems. Better tools and methods for one and two dimensional analysis of biomolecule are useful for, e.g., drug development, medical research, and the pre-diagnosis and/or diagnosis of diseases. In particular, better tools and methods are need for proteomic analysis. The present invention solves these and other problems.